1. Field of the Invention
This invention relates to novel prostaglandin derivatives, and, more especially, to the novel prostaglandin derivatives, herein deemed the prostaglandins PGB.sub.x, which are distinguishingly characterized by such exemplary in vitro and in vivo biological properties as: [1] dramatic effects on mitochondria of mammalian organisms, e.g., their ability to restore oxidative phosphorylation in degenerated mitochondria that have lost this function; [2] their protection and probable reversal of anoxic damage to mammalian brain; [3] their reversal of degenerative changes (coronary infarct) in heart; and [4] their improvement of mammalian performance for conditioned psychological tasks. The invention also relates to certain processes for the preparation of the topic prostaglandins PGB.sub.x, for example, by the base catalyzed reaction of PGB.sub.1 ; to the resolution of the reaction products into various components; and to the purification of such components into fractions exhibiting varying degrees of the aforesaid activities.
2. Description of the State of the Art
Previous studies on humans subjected to physical or psychic stress revealed common plasma increments in the level of phosphatidyl glycerol. Similar changes were found in the plasma and tissues of acceleration-stressed rats. The effects of acceleration on the plasma level of phosphatidyl glycerol could be reproduced by the injection of prostaglandin E.sub.1. In vivo experiments showed exceptionally fast turnover rates of P.sup.32 in phosphatidyl glycerol isolated from liver mitochondria of rats. These observations led to studies on the possible role of prostaglandins and phosphatidyl glycerol in phosphorylation mechanisms. When aged mitochondria were further "uncoupled" with Triton X-100 and reacted with adenosine diphosphate and P.sup.32 under conditions for oxidative phosphorylation, analysis of the reaction products by ion exchange chromatography gave increased levels of adenylic acid and inorganic phosphate. Addition of prostaglandin E.sub.1 and phosphatidyl glycerol to the reaction reversed the dephosphorylation and yielded a net increase in adenosine triphosphate correlated with a decrease in inorganic phosphate. In the presence of Triton X-100, both prostaglandin E.sub.1 and the prostaglandin B.sub.X according to the invention were equally effective in reactivating phosphorylation. In the absence of Triton X-100, prostaglandin E.sub.1 was inactive but prostaglandin B.sub.X was effective alone. Thin layer chromatography on silica of the one minute reaction products extracted by chloroform-methanol separated a radioactive phosphate labelled lipid component derived presumptively from prostaglandin B.sub.X. This implication of prostaglandin B.sub.X as a possible intermediate in mitochondrial phosphorylation offered a new probe to the mechanisms involved in the transformation of oxidative energy. Compare Polis, B.D., A. M. Pakoskey, and H. W. Shmukler, "Regeneration of Oxidative Phosphorylation In Aged Mitochondria By Prostaglandin B.sub.1 ", Proc. Nat. Acad. Sci., 63:229 (1969).
Further, the injection of various of the prostaglandins (PGE.sub.1, PGF.sub.1, PGB.sub.1 and the PGB.sub.x prostaglandins according to the invention) into rats caused changes in plasma and brain phosphatidyl glycerol and related phospholipids that mimic the changes found in accelerated rats and in the plasma of physically or psychically stressed humans. The prostaglandin effects on normal rat plasma phospholipids were abolished in the hypophysectomized rat. A similar block in phospholipid change was observed in hypophysectomized rats subjected to acceleration stress. All of the above prostaglandins caused significant increases in plasma and brain phosphatidyl glycerol. Differences were observed in the effects on other phospholipids. Thus, PGE.sub.1 decreased the total plasma phospholipid and phosphatidyl choline levels, while PGF.sub.1 increased both levels. PGE.sub.1 caused severe symptoms of lassitude and diarrhea in both normal and hypophysectomized rats. These effects were absent with the other prostaglandins. In contrast, the PGB.sub.x of the invention appeared to enhance the state of well being and lively behavior of the rat. These results, in conjunction with other previous work.sup.1 on phospholipids in stress, implicate the prostaglandins in an adaptive response to stress which involves the mobilization of energy yielding molecular components and a gearing of metabolic events for survival. See Polis et al., "Prostaglandin Induced, Stress Related, Phospholipid Changes In The Rat", Bureau of Medicine and Surgery, Work Unit No. MR005.06.01-0011B, Report No. 3, Aerospace Medical Research Department, NADC-MR-7006 (10 June 1970), NTIS Accession Document 708379, hereby expressly incorporated by reference. FNT .sup.1 Polis et al., "Effect Of Physical And Psychic Stress On Phosphatidyl Glycerol And Related Phospholipids", Biochem. Med., 2(4):286 (1969).
Moreover, on the simplistic premise that an anoxic-fatigue stress, like acceleration, could be defined in terms of energy demand under conditions of limited supply, a search has been reported for molecular probes which would reveal or reflect those regulatory mechanisms pertinent to the bioenergetic pathways involved in adaptation to stress. According to such search, it was expected that the exhaustion of adaptive events, and the onset of pathology, would be presignaled by molecular changes which might afford a biochemical index or end point to stress tolerance. It was additionally thought that such information would be useful also to amortize, pharmacologically, the energetic cost of a defensive reorganization against stress, and thereby enhance the survival of a crisis period. Compare Polis et al., "Some In Vitro And In Vivo Effects Of A New Prostaglandin Derivative" Advances Exp. Med. Biol., 33, 213 (1972), hereby expressly incorporated by reference and relied upon, and wherein it is indicated:
Experiments with isolated particulate fractions from animal cells revealed marked changes in a specific phospholipid identified as phosphatidyl glycerol (G) that followed exposure to an acute stress like acceleration or a longer termed degenerative stress like X-irradiation. These stress induced changes in the phospholipid composition of tissues and their correlation with comparable changes in plasma phospholipds of the rat suggested an approach to stress induced chemical changes in humans.
The effects of both physical and psychic stress on human plasma phospholipids are shown in FIG. 11 infra. These are portrayed as three dimensional plots of the means.+-.two standard errors for G, phosphatidic acid (P) and phosphatidyl ethanolamine (PE). It is evident that in all the populations exposed to the various stresses of acceleration, sleep deprivation, combat flying, or the stress accompanying schizophrenia, there was a significant increase in G over the controls. Variations in other phospholipids made possible the statistical discrimination of stressed populations from each other.
In all the stress reactions studied, G was unique in the consistently elevated plasma levels which were common to all the stresses. In contrast, other phospholipids showed variable changes which facilitated a molecular characterization of the stress. These concentration shifts in individual phospholipids were not a direct consequence of variations in the total phospholipid content. Both increments and decrements of specific phospholipids were observed with no changes or even opposing changes in the levels of the total phospholipid content. Whether this represented a concentration effect in the output of a major regulatory factor or whether each phospholipid was uniquely controlled, the results implied the action of some brain centers which interpreted sensory inputs as "threats to survival" and in reacting, mobilized the phospholipids. That this hypothesis had some merit was indicated by the release of G from the brains of stressed humans.
Also shown were the results of collaborative studies on the differences in G between jugular venous and femoral arterial blood plasma from subjects under control conditions and after acceleration to grayout. It is apparent that there is indeed a significant release of G from the brains of the subjects after acceleration. The singularity of G is emphasized by the failure of all other phospholipid species to show any significant concentration change across the human brain after grayout.
Some indications of triggering factors for the phospholipid changes in stress can be obtained from the fact that the injection of various prostaglandins into rats caused major changes in G and lesser changes in other phospholipids that mimic the results obtained in stressed rats and humans. The increases in plasma G in the rat were accompanied by elevations of brain G. Although all four of the prostaglandins shown in FIG. 1, including the prostaglandins PGB.sub.x according to the invention, caused elevations of plasma G, they differed in their effects on other phospholipids. Prostaglandin E.sub.1 (PGE.sub.1) caused a significant decrease in lecithin (Le) and total phospholipid while Prostaglandin F.sub.1.alpha. (PGF.sub.1.alpha.) caused an increase in Le and total phospholipid. The changes in total phospholipid were nonsignificant for PGB.sub.1 and the topic PGB.sub.x and the variation in Le less marked. Variable effects on the concentration changes of P were observed with the different prostaglandins. The greatest change was obtained with PGE.sub.1. There also were marked differences in observable physiological response. PGE.sub.1 injection was followed by severe lassitude and diarrhea so that the rats appeared visibly ill. This response was absent with the other prostaglandins. With the subject PGB.sub.x, the rats even appeared more lively and excitable.